1. Immunoprecipitation

2. Definition of Immunology
The study of the physiological mechanisms which enable the body to recognize materials as foreign and to neutralize, eliminate, or metabolize them without injury to the host tissue.

3. Overview of Immunodiagnostic Studies
Antigen-antibody reactions for diagnosis of:
infectious disease,
autoimmune disorders,
immune allergies,
and neoplastic disease.
These modalities also test for:
blood groups and types,
tissue and graft transplant matching.

4. Types of Tests
Many methods of varying sophistication are used for immunodiagnostic studies

5. Types of Tests Tests for Visible Change Observable Reaction
Name of Test
Treponemal, heterophile, and cold agglutinin antibodies
Clumping
Particulate antigen reacts with corresponding antibody; antigen may be in form of RBCs (hemagglutination, latex, or charcoal coated with antigen).
Agglutination
Fungal antibodies, food poisoning, Quantitative immunoglobulins IgA, IgG
Precipitates
Soluble antigen reacts with corresponding antibody by ID or count.
Precipitation
Viral antibodies
Complement activation, hemolysis
Competition between two antigen-antibody systems (test and indicator systems)
Complement fixation (CF)
Antinuclear antibodies (ANAs); antimitochondrial antibodies (AMAs)
Visible microscopic fluorescence
Fluorescent-tagged antibody reacts with antigen-antibody complex in the presence of ultraviolet light.
Immunofluorescence
Hepatitis and human immunodeficiency virus (HIV) (screening)
Color change indicates enzyme substrate reaction.
Indirect EIA for quantification of an antigen or antibody enzyme and substrate
Enzyme-linked immunosorbent assay (ELISA)
Confirms HIV-1
Detection of antibodies of specific mobility
Electrophoresis separation of antigen subspecies
Immunoblot (eg, Western blot [WB])
Quantitative immunoglobulins IgA, IgM, C-reactive protein, anti-streptolysin O recorded in mg/dL or IU/mL
Light scatter proportionately increases as numbered size of immune complexes increases.
Measures either antigen or antibody in solution through the scattering of a light beam; antibody reagent used to detect antigen IgA, IgG, IgM; concurrent controls are run to establish amount of background scatter in reagents and test samples.
Rate nephelometry

6. Antigen-antibody interaction
Antigens are substances that stimulate and subsequently react with the products of an immune response. They may be:
enzymes,
toxins,
microorganisms (eg, bacterial, viral, parasitic, fungal),
tumors,
or autoimmune factors.
Antibodies are proteins produced by the body's immune system in response to an antigen or antigens. The antigen-antibody response is the body's natural defense against invading organisms.

7. Structure of an antibody

8. Antibody Structure V Light Chain Light Chain Heavy Chains Constant SS
Antigen binding site V SS SS
Light
Chain
Light Chain
Constant
Heavy Chains

9. Classes of antibodies
IgG - A monomer - Most abundant antibody in blood. IgG easily leaves the circulatory system to fight infection and crosses the placenta conferring passive immunity to a fetus.
IgD - A monomer - Found on the surface of B cells probably allowing recognition of antigens thus triggering differentiation into plasma and memory B cells
IgE - A monomer - The least common antibody. The tails attach to mast cells and basophils. When antigens bind, they signal release of histamine.
IgA - A dimer - Produced by cells in the mucus membranes to prevent attachment of pathogens. IgA is also found in many body secretions including milk.
IgM - A pentamer - First antibody to appear following exposure to an antigen. Because it declines rapidly in the blood, high IgM levels indicate a current infection.

10. Three Distinct Phases of Antigen/Antibody Reactions
Primary Phenomenon –
involves the combination of an individual binding site on an antibody molecule with a single epitope or determinant site on an antigen
Secondary Phenomenon –
Lattice formation include precipitation, agglutination and complement fixation
Tertiary Phenomenon –
Detected by affect on tissues or cells, Inflammation, phagocytosis, deposition of immune complexes, immune adherence and chemotaxis

11. Detection of antigen-antibody interactions:
Primary Phenomenon e.g.
Enzyme linked immuono sorben assay (ELISA)
Immuno flurescent antibody technique (IFAT)
Radio immuno assay (RIA)
Secondary Phenomenon : e.g.
Agglutination tests
Complement fixation tests (CFT)
Precipitation tests
Tertiary Phenomenon : e.g.
Reaction not visible, detected by affect of reaction on tissues or cells

12. Antigen Binding
Antigen 2
Antigen 1
Antigen 3
Variable
Light
Heavy

13. Ag-Ab interactions Bonds: Hydrogen Ionic Hydrophobic interactions
Van der Waals forces
Each bond is weak; many are strong
To “hold” they must be close and complementary

14. Non-covalent binding

15. Features of the Antigen-Antibody Interaction
Reversibility
Non-covalent Interactions
Affinity
Measure of the strength of the binding
Ease of association or dissociation
Avidity
Increase in affinity due to multivalent binding
The summation of multiple affinities

16. Binding of the epitope in the antigen binding site
POOR FIT
GOOD FIT
antibody combining site
antigen determinant
high attraction
low repulsion
high repulsion
low attraction
High Affinity
Low Affinity

17. Antibody Affinity
Affinity – the strength of the total noncovalent interactions between a single Ag-binding site on an Ab and a single epitope
Low affinity Abs bind Ag weakly and dissociate readily
High affinity Abs bind Ag more tightly and remain bound longer
Ag + Ab
Ab-Ag k1
k-1
K1 = forward (association) constant
K-1 = reverse (dissociation) constant

18. Antibody Affinity
K can be determined using equilibrium dialysis

19. K determined by equilibrium dialysis

20. Antibody Avidity
Avidity – the strength of the total noncovalent interactions between a multivalent Ab and Ag
The avidity of an antibody is a better measure of its binding capacity within biological systems than is the affinity of its individual binding sites
High avidity can compensate for low affinity; eg. pentameric IgM often has lower affinity than IgG but its higher avidity (due to presence of 10 binding sites) may enable it to bind Ag more effectively

21. Antibody Avidity Y
Keq =
104
Affinity Y
106
Avidity Y
1010
Avidity

22. Effect of multivalent interactions
antibody
Fab
IgG
IgG
IgM
effective antibody valence 1 1 2
up to 10
antigen valence 1 1 n n
equilibrium constant (L/M) 10 4 10 4 10 7 10 11
advantage of multi-valence - - 10 3
-fold 10 7
-fold
definition of bindng
affinity
affinity
avidity
avidity
intrinsic affinity
functional affinity

23. Factors Affecting Measurement of Ag/Ab Reactions
Ab excess
Ag excess
Affinity
Avidity
Equivalence – Lattice formation
Ag:Ab ratio
Physical form of Ag

24. Tests Based on Ag/Ab Reactions
All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes
All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

25. Immunoprecipitation
Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody that specifically binds to that particular antigen. This process can be used to isolate and concentrate a particular antigen from a sample containing many thousands of different antigens.

26. Immunoprecipitation tests
The antigen and antibody are in soluble form
Combine to form a visible precipitate
Complete precipitation occurs at certain Ab/Ag concentrations
Antigen
Antibody

27. Precipitation Curve
Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative
Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative
Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

28. Precipitation Curve

29. Precipitation Reactions
Ab + soluble Ag
Lattice
Precipitate
Precipitation techniques
Tube precipitation test
Gel diffusion
Single radial
Double
Immunoelectrophoresis
Countercurrent electrophoresis (CEP),

30. Tube precipitation test

31. Gel diffusion Immunodiffusion tests
Antigen and antibody placed in separate wells cut in gel
Solutions diffuse and meet between the wells
Results in line of precipitation at zone of optimal proportion

32. Double Diffusion
Note: A line of precipitation has formed between the center well and wells 3 & 5. This indicates there is antigen/antibody specificity between the center well and these two wells.
Usually a known antigen or known antibody is placed in the center and test serum is placed in the peripherial wells.
Antigens and antibodies will diffuse and at some point optimal concentrations will occur and if the antigen is specific for the antibody a precipitate line will form.
Example: Has this patient ever had rubella, or diptheria? If they have their serum will contain antibodies against the disease.
Put patient serum in the center. Put the disease agents (antigens) in wells 1 – 5, and allow to diffuse. A precipitation line between wells indicates that the patient has had that disease

33. Double Diffusion Ab and Ag diffuse towards each other
Used for determining antigenic relationship between substances
Identity
Non-identity
Partial identity

34. Double Diffusion 1 2 Ab

35. Double Diffusion

36. Identity
If wells 1 and 2 contain two identical antigens and well 3 has antibodies to that antigen, a reaction of identity results as all antibodies react with what is in both antigen wells
indicating that the contents of both wells are closely related. X X
Anti X Y
Anti X

37. Identity: What You Really See

38. Double Diffusion

39. Non-identity
If wells 1 and 2 contain two separate antigens and well 3 has antibodies to both antigens, a reaction of non-identity results
indicating that either X and Y are different or that well 3 has a mixture of two antibodies in it. X Y
Anti X Y
Anti X
Anti Y

40. Non-identity: What You Really See

41. Double Diffusion

42. Partial Identity
If wells 1 and 2 contain two separate antigens and well 3 has antibodies to both antigens, a reaction of partial identity may result if all antibodies react with X, but only some react with Y.
indicating that X and Y are related in some way. X Y
Anti X Y
Anti X
Anti Y

43. Non-identity: What You Really See

44. Radial immunodiffusion test
Radial immunodiffusion test is quantitative
Antibody is added to liquid agar that is allowed to harden
Created a uniform antibody concentration
Antigen added to wells cut in gel
Diffusion outward forms concentration gradient
Ring forms at antigen-antibody precipitation
Standards can be used to construct standard curve to establish concentration

45. Single radial immunodiffusionAg

46. Single radial immunodiffusion

47. Single radial immunodiffusion

48. Single radial immunodiffusion
Interpretation
Diameter of ring is proportional to the concentration
Quantitative
Ig levels Ag
Ab in gel
Ag Concentration
Diameter2

49. Single radial immunodiffusion

50. Single radial immunodiffusion
Exercise 1

51. Materials Standard Single radial immunodifusion plates
Moisture chamber
Micropipettes

52. Specimen
Collect whole blood without anticoagulant and allow to clot at room temperature. Separate serum by centrifugation within 2-3 hours after collection.

53. Method
Remove plates from refrigerator to room temperature approximately 30 minutes before filling wells. Do not open bag until ready for use.
If excess moisture is present, remove plate from its bag and remove cover until evaporation has dried the surface and wells. Replace cover until used.
For best results, three wells should be filled with reference sera for each plate. Location of each should be noted.

54. Preparing the standards (serial dilution)
Label four microtest tubes: 1:2, 1:4, 1:8, and 1:16.
Using a micropipette, add 50 microliters of Buffer to each tube.
With a fresh pipet tip, add 50 microliters of “Standard” to the tube labeled 1:2, Mix. Transfer 50 microliters of the 1:2 dilution to the tube labeled 1:4, Mix, and so on.

55. Performance of Test
Deliver specimen or standard (5 µl) to well by placing the pipette tip at the bottom of the well. Allow the well to fill to the top of the agar surface. Avoid bubbles to ensure proper volume and diffusion of sample. Visualization may be aided by placing the plate on dark background.
Mark time of completion on plate cover and replace cover.
Replace plate in bag and reseal carefully.
Incubate plates upright on a flat surface at room temperature (20 to 24oC) over 48 hours for End Point readings.

56. Calibration Curve
Using the reference sera determine their ring diameters to the nearest 0.1mm.
Using regular graph paper plot the concentration on the X axis and the zone diameters squared on the Y axis for each protein for End point readings.
Draw a straight line of "best fit" between the four points.

57. Results
Measure diameters of precipitin zones to within 0.1mm. Variations in incubation times of more than 30 minutes will produce changes in diameters, especially those at higher levels of antigen except when plates have reached endpoint.
Determine the concentration of each unknown or specimen protein from the reference curve find the corresponding concentration.

58. Results
Ag Concentration
Diameter2